Osteoprotegerin binding proteins

ABSTRACT

A novel polypeptide, osteoprotegerin binding protein, involved in osteolcast maturation has been identified based upon its affinity for osteoprotegerin. Nucleic acid sequences encoding the polypeptide, or a fragment, analog or derivative thereof, vectors and host cells for production, methods of preparing osteoprotegerin binding protein, and binding assays are also described. Compositions and methods for the treatment of bone diseases such as osteoporosis, bone loss due to arthritis or metastasis, hypercalcemia, and Paget&#39;s disease are also provided.

FIELD OF THE INVENTION

[0001] The present invention relates to polypeptides which are involvedin osteoclast differentiation. More particularly, the invention relatesto osteoprotegerin binding proteins, nucleic acids encoding theproteins, expression vectors and host cells for production of theproteins, and binding assays. Compositions and methods for the treatmentof bone diseases, such as osteoporosis, bone loss from arthritis,Paget's disease, and hypercalcemia, are also described.

BACKGROUND OF THE INVENTION

[0002] Living bone tissue exhibits a dynamic equilibrium betweendeposition and resorption of bone. These processes are mediatedprimarily by two cell types: osteoblasts, which secrete molecules thatcomprise the organic matrix of bone; and osteoclasts, which promotedissolution of the bone matrix and solubilization of bone salts. Inyoung individuals with growing bone, the rate of bone deposition exceedsthe rate of bone resorption, while in older individuals the rate ofresorption can exceed deposition. In the latter situation, the increasedbreakdown of bone leads to reduced bone mass and strength, increasedrisk of fractures, and slow or incomplete repair of broken bones.

[0003] Osteoclasts are large phagocytic mutinucleated cells which areformed from hematopoietic precursor cells in the bone marrow. Althoughthe growth and formation of mature functional osteoclasts is not wellunderstood, it is thought that osteoclasts mature along themonocyte/macrophage cell lineage in response to exposure to variousgrowth-promoting factors. Early development of bone marrow precursorcells to preosteoclasts are believed to mediated by soluble factors suchas tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β),interleukin-1 (IL-1), interleukin-4 (IL-4), interleukin-6 (IL-6), andleukemia inhibitory factor (LIF). In culture, preosteoclasts are formedin the presence of added macrophage colony stimualting factor (M-CSF).These factors act primarily in early steps of osteoclast development.The involvement of polypeptide factors in terminal stages of osteoclastformation has not been extensively reported. It has been reported,however, that parathyroid hormone stimulates the formation and activityof osteoclasts and that calcitonin has the opposite effect, although toa lesser extent.

[0004] Recently, a new polypeptide factor, termed osteoprotegerin (OPG),has been described which negatively regulated formation of osteoclastsin vitro and in vivo (see co-owned and co-pending U.S. Ser. Nos.08/577,788 filed Dec. 22, 1995, 08/706,945 filed Sep. 3, 1996, and08/771,777, filed Dec. 20, 1996, hereby incorporated by reference; andPCT Application No. WO96/26271). OPG dramatically increased the bonedensity in transgenic mice expressing the OPG polypeptide and reducedthe extent of bone loss when administered to ovariectomized rats. Ananalysis of OPG activity in in vitro osteoclast formation revealed thatOPG does not interfere with the growth and differentiation ofmonocyte/macrophage precursors, but more likely blocks thedifferentiation of osteoclasts from monocyte/macrophage precursors. ThusOPG appears to have specificity in regulating the extent of osteoclastformation.

[0005] OPG comprises two polypeptide domains having different structuraland functional properties. The amino-terminal domain spanning aboutresidues 22-194 of the full-length polypeptide (the N-terminalmethionine is designated residue 1) shows homology to other members ofthe tumor necrosis factor receptor (TNFR) family, especially TNFR-2,through conservation of cysteine rich domains characteristic of TNFRfamily members. The carboxy terminal domain spanning residues 194-401has no significant homology to any known sequences. Unlike a number ofother TNFR family members, OPG appears to be exclusively a secretedprotein and does not appear to be synthesized as a membrane associatedform.

[0006] Based upon its activity as a negative regulator of osteoclastformation, it is postulated that OPG may bind to a polypeptide factorinvolved in osteoclast differentiation and thereby block one or moreterminal steps leading to formation of a mature osteoclast.

[0007] It is therefore an object of the invention to identifypolypeptides which interact with OPG. Said polypeptides may play a rolein osteoclast maturation and may be useful in the treatment of bonediseases.

SUMMARY OF THE INVENTION

[0008] A novel member of the tumor necrosis factor family has beenidentified from a murine cDNA library expressed in COS cells screenedusing a recombinant OPG-Fc fusion protein as an affinity probe. The newpolypeptide is a transmembrane OPG binding protein which is predicted tobe 316 amino acids in length, and has an amino terminal cytoplasmicdomain, a transmembrane doman, and a carboxy terminal extracellulardomain. OPG binding proteins of the invention may be membrane-associatedor may be in soluble form.

[0009] The invention provides for nucleic acids encoding an OPG bindingprotein, vectors and host cells expressing the polypeptide, and methodfor producing recombinant OPG binding protein. Antibodies or fragmentsthereof which specifically bind OPG binding protein are also provided.

[0010] OPG binding proteins may be used in assays to quantitate OPGlevels in biological samples, identify cells and tissues that displayOPG binding protein, and identify new OPG and OPG binding protein familymembers. Methods of identifying compounds which interact with OPGbinding protein are also provided. Such compounds include nucleic acids,peptides, proteins, carbohydrates, lipids or small molecular weightorganic molecules and may act either as agonists or antagonists of OPGbinding protein activity.

[0011] OPG binding proteins are involved in osteoclast differentiationand the level of osteoclast activity in turn modulates bone resorption.OPG binding protein agonists and antagonists modulate osteoclastformation and bone resorption and may be used to treat bone diseasescharacterized by changes in bone resorption, such as osteoporosis,hypercalcemia, bone loss due to arthritis metastasis, immobilization orperiodontal disease, Paget's disease, osteopetrosis, prostheticloosening and the like. Pharmaceutical compositions comprising OPGbinding proteins and OPG binding protein agonists and antagonists arealso encompassed by the invention.

DESCRIPTION OF THE FIGURES

[0012]FIG. 1. Structure and sequence of the 32D-F3 insert encoding OPGbinding protein. Predicted transmembrane domain and sites forasparagine-linked carbohydrate chains are underlined.

[0013]FIG. 2. OPG binding protein expression in COS-7 cells transfectedwith pcDNA/32D-F3. Cells were lipofected with pcDNA/32D-F3 DNA, theassayed for binding to either goat anti-human IgG1 alkaline phosphataseconjugate (secondary alone), human OPG[22-201]-Fc plus secondary(OPG-Fc), or a chimeric ATAR extracellular domain-Fc fusion protein(sATAR-Fc). ATAR is a new member of the TNFR superfamily, and thesATAR-Fc fusion protein serves as a control for both human IgG1 Fcdomain binding, and generic TNFR releated protein, binding to 32D cellsurface molecules.

[0014]FIG. 3. Expression of OPG binding protein in human tissues.Northern blot analysis of human tissue mRNA (Clontech) using aradiolabeled 32D-F3 derived hybridization probe. Relative molecular massis indicated at the left in kilobase pairs (kb). Arrowhead on right sideindicates the migration of an approximately 2.5 kb transcript detectedin lymph node mRNA. A very faint band of the same mass is also detectedin fetal liver.

[0015]FIG. 4. Structure and sequence of the pcDNA/hu OPGbp 1.1 insertencoding the human OPG binding protein. The predicted transmembranedomain and site for asparagine-linked charbohydrate chains areunderlined.

[0016]FIG. 5. Stimulation of osteoclast development in vitro from bonemarrow macrophage and ST2 cell cocultures treated with recombinantmurine OPG binding protein [158-316]. Cultures were treated with varyingconcentrations of murine OPG binding protein ranging from 1.6 to 500ng/ml. After 8-10 days, cultures were lysed, and TRAP activity wasmeasured by solution assay. In addition, some cultures weresimultaneously treated with 1, 10, 100, 500, and 1000 ng/ml ofrecombinant murine OPG [22-401]-Fc protein. Murine OPG binding proteininduces a dose-dependent stimulation in osteoclast formation, whereasOPG [22-401]-Fc inhibits osteoclast formation.

[0017]FIG. 6. Stimulation of osteoclast development from bone marrowprecursors in vitro in the presence of M-CSF and murine OPG bindingprotein [158-316]. Mouse bone marrow was harvested, and cultured in thepresence 250, 500, 1000, and 2000 U/ml of M-CSF. Varying concentrationsof OPG binding protein [158-316], ranging from 1.6 to 500 ng/ml, wereadded to these same cultures. Osteoclast development was measured byTRAP solution assay.

[0018]FIG. 7. Osteoclasts derived from bone marrow cells in the presenceof both M-CSF and OPG binding protein [158-316] resorb bone in vitro.Bone marrow cells treated with either M-CSF, OPG binding protein, orwith both factors combined, were plated onto bone slices in culturewells, and were allowed to develop into mature osteoclasts. Theresulting cultures were then stained with Toluidine Blue (left column),or histochemically to detect TRAP enzyme activity (right column). Incultures receiving both factors, mature osteoclasts were formed thatwere capable of eroding bone as judged by the presence of blue stainedpits on the bone surface. This correlated with the presence of multiplelarge, multinucleated, TRAP positive cells.

[0019]FIG. 8. Graph showing the whole blood ionized calcium (iCa) levelsfrom mice injected with OPG binding protein, 51 hours after the firstinjection, and in mice also receiving concurrent OPG administration. OPGbinding protein significantly and dose dependently increased iCa levels.OPG (1 mg/kg/day) completely blocked the increase in iCa at a dose ofOPG binding protein of 5 ug/day, and partially blocked the increase at adose of OPG binding protein of 25 ug/day. (*), different to vehicletreated control (p<0.05). (#), OPG treated iCa level significantlydifferent to level in mice receiving that dose of OPG binding proteinalone (p<0.05).

[0020]FIG. 9. Radiographs of the left femur and tibia in mice treatedwith 0, 5, 25 or 100 ug/day of OPG binding protein for 3.5 days. Thereis a dose dependent decrease in bone density evident most clearly in theproximal tibial metaphysis of these mice, and that is profound at a doseof 100 ug/day.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The invention provides for a polypeptide referred to as an OPGbinding protein, which specficially binds OPG and is involved inosteoclast differentiation. A cDNA clone encoding the murine form of thepolypeptide was identified from a library prepared from a mousemyelomonocytic cell line 32-D and transfected into COS cells.Transfectants were screened for their ability to bind to anOPG[22-201]-Fc fusion polypeptide (Example 1). The nucleic acid sequencerevealed that OPG binding protein is a novel member of the TNF familyand is most closely related to AGP-1, a polypeptide previously describedin co-owned and co-pending U.S. Ser. No. 08/660,562, filed Jun. 7, 1996.(A polypeptide identical to AGP-1 and designated TRAIL is described inWiley et al. Immunity 3, 673-682 (1995)). OPG binding protein ispredicted to be a type II transmembrane protein having a cytoplamsicdomain at the amino terminus, a transmembrane domain, and a carboxyterminal extracellular domain (FIG. 1). The amino terminal cytoplasmicdomain spans about residues 1-48, the transmembrane domain spans aboutresidues 49-69 and the extracellular domain spans about residues 70-316as shown in FIG. 1 (SEQ ID NO:_______). The membrane-associated proteinspecifically binds OPG (FIG. 2). Thus OPG binding protein and OPG sharemany characteristics of a receptor-ligand pair although it is possiblethat other naturally-occurring receptors for OPG binding protein exist.

[0022] A DNA clone encoding human OPG binding protein was isolated froma lymph node cDNA library. The human sequence (FIG. 4) is homologous tothe murine sequence. Purified soluble murine OPG binding proteinstimulated osteoclast formation in vitro and induced hypercalcemia andbone resorption in vivo.

[0023] OPG binding protein refers to a polypeptide having an amino acidsequence of mammalian OPG binding protein, or a fragment, analog, orderivative thereof, and having at least the activity of binding OPG. Inpreferred embodiments, OPG binding protein is of murine or human origin.In another embodiment, OPG binding protein is a soluble protein having,in one form, an isolated extracellular domain separate from cytoplasmicand transmembrane domains. OPG binding protein is involved in osteoclastdifferentiation and in the rate and extent of bone resorption, and wasfound to stimulate osteoclast formation and stimulate bone resorption.

[0024] Nucleic Acids

[0025] The invention provides for isolated nucleic acids encoding OPGbinding proteins. As used herein, the term nucleic acid comprises cDNA,genomic DNA, wholly or partially synthetic DNA, and RNA. The nucleicacids of the invention are selected from the group consisting of:

[0026] a) the nucleic acids as shown in FIG. 1 (SEQ ID NO:______) andFIG. 4 (SEQ ID NO:______);

[0027] b) nucleic acids which hybridize to the polypeptide codingregions of the nucleic acids shown in FIG. 1 (SEQ ID NO:______) and FIG.4 (SEQ ID NO:______); and remain hybridized to the nucleic acids underhigh stringency conditions; and

[0028] c) nucleic acids which are degenerate to the nucleic acids of (a)or (b).

[0029] Nucleic acid hybridizations typically involve a multi-stepprocess comprising a first hybridization step to form nucleic acidduplexes from single strands followed by a second hybridization stepcarried out under more stringent conditions to selectively retainnucleic acid duplexes having the desired homology. The conditions of thefirst hybridization step are generally not crucial, provided they arenot of higher stringency than the second hybridization step. Generally,the second hybridization is carried out under conditions of highstringency, wherein “high stringency” conditions refers to conditions oftemperature and salt which are about 12-20° C. below the meltingtemperature (T_(m)) of a perfect hybrid of part or all of thecomplementary strands corresponding to FIG. 1 (SEQ. ID. NO:______) andFIG. 4 (SEQ ID NO:______). In one embodiment, “high stringency”conditions refer to conditions of about 65° C. and not more than about1M Na+. It is understood that salt concentration, temperature and/orlength of incubation may be varied in either the first or secondhybridization steps such that one obtains the hybridizing nucleic acidmolecules according to the invention. Conditions for hybridization ofnucleic acids and calculations of T_(m) for nucleic acid hybrids aredescribed in Sambrook et al. Molecular Cloning: A Laboratory Manual ColdSpring Harbor Laboratory Press, New York (1989).

[0030] The nucleic acids of the invention may hybridize to part or allof the polypeptide coding regions of OPG binding protein as shown inFIG. 1 (SEQ ID NO:______) and FIG. 4 (SEQ ID NO:______); and thereforemay be truncations or extensions of the nucleic acid sequences showntherein. Truncated or extended nucleic acids are encompassed by theinvention provided that they retain at least the property of bindingOPG. In one embodiment, the nucleic acid will encode a polypeptide of atleast about 10 amino acids. In another embodiment, the nucleic acid willencode a polypeptide of at least about 20 amino acids. In yet anotherembodiment, the nucleic acid will encode a polypeptide of at least about50 amino acids. The hybridizing nucleic acids may also include noncodingsequences located 5′ and/or 3′ to the OPG binding protein codingregions. Noncoding sequences include regulatory regions involved inexpression of OPG binding protein, such as promoters, enhancer regions,translational initiation sites, transcription termination sites and thelike.

[0031] In preferred embodiments, the nucleic acids of the inventionencode mouse or human OPG binding protein. Nucleic acids may encode amembrane bound form of OPG binding protein or soluble forms which lack afunctional transmembrane region. The predicted transmembrane region formurine OPG binding protein includes amino acid residues 49-69 inclusiveas shown in FIG. 1 (SEQ. ID. NO:______). The predicted transmembraneregion for human OPG binding protein includes residues 49-69 as shown inFIG. 4 (SEQ ID NO:______). Substitutions which replace hydrophobic aminoacid residues in this region with neutral or hydrophilic amino acidresidues would be expected to disrupt membrane association and result insoluble OPG binding protein. In addition, deletions of part or all thetransmembrane region would also be expected to produce soluble forms ofOPG binding protein. Nucleic acids encoding amino acid residues 70-316as shown in FIG. 1 (SEQ ID NO:______), or fragments and analogs thereof,encompass soluble OPG binding proteins.

[0032] Nucleic acids encoding truncated forms of soluble human OPGbinding proteins are also included. Soluble forms include residues69-317 as shown in FIG. 4 (SEQ ID NO:______) and truncations thereof. Inone embodiment, N-terminal truncations generate polypeptides fromresidues, 70-317, 71-317, 72-317, and so forth. In another embodiment,nucleic acids encode soluble OPGbp comprising residues 69-317 andN-terminal truncations thereof up to OPGbp [158-317], or alternatively,up to OPGbp [166-317].

[0033] Plasmid phuOPGbp 1.1 in E. coli strain DH10 encoding human OPGbinding protein was deposited with the American Type Culture Collection,Rockville, Md. on Jun. 13, 1997.

[0034] Nucleic acid sequences of the invention may be used for thedetection of sequences encoding OPG binding protein in biologicalsamples. In particular, the sequences may be used to screen cDNA andgenomic libraries for related OPG binding protein sequences, especiallythose from other species. The nucleic acids are also useful formodulating levels of OPG binding protein by anti-sense technology or invivo gene expression. Development of transgenic animals expressing OPGbinding protein is useful for production of the polypeptide and for thestudy of in vivo biological activity.

[0035] Vectors and Host Cells

[0036] The nucleic acids of the invention will be linked with DNAsequences so as to express biologically active OPG binding protein.Sequences required for expression are known to those skilled in the artand include promoters and enhancer sequences for initiation of RNAsynthesis, transcription termination sites, ribosome binding sites forthe initiation of protein synthesis, and leader sequences for secretion.Sequences directing expression and secretion of OPG binding protein maybe homologous, i.e., the sequences are identical or similar to thosesequences in the genome involved in OPG binding protein expression andsecretion, or they may be heterologous. A variety of plasmid vectors areavailable for expressing OPG binding protein in host cells (see, forexample, Methods in Enzymology v. 185, Goeddel, D. V. ed., AcademicPress (1990)). For expression in mammalian host cells, a preferredembodiment is plasmid pDSRα described in PCT Application No. 90/14363.For expression in bacterial host cells, preferred embodiments includeplasmids harboring the lux promoter (see co-owned and co-pending U.S.Ser. No. 08/577,778, filed Dec. 22, 1995). In addition, vectors areavailable for the tissue-specific expression of OPG binding protein intransgenic animals. Retroviral and adenovirus-based gene transfervectors may also be used for the expression of OPG binding protein inhuman cells for in vivo therapy (see PCT Application No. 86/00922).

[0037] Procaryotic and eucaryotic host cells expressing OPG bindingprotein are also provided by the invention. Host cells includebacterial, yeast, plant, insect or mammalian cells. OPG binding proteinmay also be produced in transgenic animals such as mice or goats.Plasmids and vectors containing the nucleic acids of the invention areintroduced into appropriate host cells using transfection ortransformation techniques known to one skilled in the art. Host cellsmay contain DNA sequences encoding OPG binding protein as shown in FIG.1 or a portion thereof, such as the extracellular domain or thecytoplasmic domain. Nucleic acids encoding OPG binding proteins may bemodified by substitution of codons which allow for optimal expression ina given host. At least some of the codons may be so-called preferencecodons which do not alter the amino acid sequence and are frequentlyfound in genes that are highly expressed. However, it is understood thatcodon alterations to optimize expression are not restricted to theintroduction of preference codons. Examples of preferred mammalian hostcells for OPG binding protein expression include, but are not limited toCOS, CHOd-, 293 and 3T3 cells. A preferred bacterial host cell isEscherichia coli.

[0038] Polypeptides

[0039] The invention also provides OPG binding protein as the product ofprocaryotic or eucaryotic expression of an exogenous DNA sequence, i.e.,OPG binding protein is recombinant OPG binding protein. Exogenous DNAsequences include cDNA, genomic DNA and synthetic DNA sequences. OPGbinding protein may be the product of bacterial, yeast, plant, insect ormammalian cells expression, or from cell-free translation systems. OPGbinding protein produced in bacterial cells will have an N-terminalmethionine residue. The invention also provides for a process ofproducing OPG binding protein comprising growing procaryotic oreucaryotic host cells transformed or transfected with nucleic acidsencoding OPG binding protein and isolating polypeptide expressionproducts of the nucleic acids.

[0040] Polypeptides which are mamalian OPG binding proteins or arefragments, analogs or derivatives thereof are encompassed by theinvention. In a preferred embodiment, the OPG binding protein is humanOPG binding protein. A fragment of OPG binding protein refers to apolypeptide having a deletion of one or more amino acids such that theresulting polypeptide has at least the property of binding OPG. Saidfragments will have deletions originating from the amino terminal end,the carboxy terminal end, and internal regions of the polypeptide.Fragments of OPG binding protein are at least about ten amino acids, atleast about 20 amino acids, or at least about 50 amino acids in length.In preferred embodiments, OPG binding protein will have a deletion ofone or more amino acids from the transmembrane region (amino acidresidues 49-69 as shown in FIG. 1), or, alternatively, one or more aminoacids from the amino-terminus up to and/or including the transmembraneregion (amino acid residues 1-49 as shown in FIG. 1). In anotherembodiment, OPG binding protein is a soluble protein comprising, forexample, amino acid residues 69-316, or 70-316, or N-terminal orC-terminal truncated forms thereof, which retain OPG binding activity.OPG binding protein is also a human soluble protein as shown in FIG. 4comprising residues 69-317 as shown in FIG. 4 and N-terminal truncatedforms thereof, e.g., 70-517, 71-517, 71-317, 72-317 and so forth. In apreferred embodiment, the soluble human OPG binding protein comprisingresidues 69-317 and N-terminal truncation thereof up to OPGbp [158-317],or alternatively up to OPG [166-317].

[0041] An analog of an OPG binding protein refers to a polypeptidehaving a substitution or addition of one or more amino acids such thatthe resulting polypeptide has at least the property of binding OPG. Saidanalogs will have substitutions or additions at any place along thepolypeptide. Preferred analogs include those of soluble OPG bindingproteins. Fragments or analogs may be naturally occurring, such as apolypeptide product of an allelic variant or a mRNA splice variant, orthey may be constructed using techniques available to one skilled in theart for manipulating and synthesizing nucleic acids. The polypeptidesmay or may not have an amino terminal methionine residue

[0042] Also included in the invention are derivatives of OPG bindingprotein which are polypeptides that have undergone post-translationalmodifications (e.g., addition of N-linked or O-linked carbohydratechains, processing of N-terminal or C-terminal ends), attachment ofchemical moieties to the amino acid backbone, chemical modifications ofN-linked or O-linked carbohydrate chains, and addition of an N-terminalmethionine residue as a result of procaryotic host cell expression. Inparticular, chemically modified derivatives of OPG binding protein whichprovide additional advantages such as increased stability, longercirculating time, or decreased immunogenicity are contemplated. Ofparticular use is modification with water soluble polymers, such aspolyethylene glycol and derivatives thereof (see for example U.S. Pat.No. 4,179,337). The chemical moieties for derivitization may be selectedfrom water soluble polymers such as polyethylene glycol, ethyleneglycol/propylene glycol copolymers, carboxymethylcellulose, dextran,polyvinyl alcohol and the like. The polypeptides may be modified atrandom positions within the molecule, or at predetermined positionswithin the molecule and may include one, two, three or more attachedchemical moieties. Polypeptides may also be modified at pre-determinedpositions in the polypeptide, such as at the amino terminus, or at aselected lysine or arginine residue within the polypeptide. Otherchemical modificaitons provided include a detectable label, such as anenzymatic, fluorescent, isotopic or affinity label to allow fordetection and isolation of the protein.

[0043] OPG binding protein chimeras comprising part or all of an OPGbinding protein amino acid sequence fused to a heterologous amino acidsequence are also included. The heterologous sequence may be anysequence which allows the resulting fusion protein to retain the atleast the activity of binding OPG. In a preferred embodiment, thecarboxy terminal extracellular domain of OPG binding protein is fused toa heterologous sequence. Such sequences include heterologous cytoplasmicdomains that allow for alternative intracellular signalling events,sequences which promote oligomerization such as the Fc region of IgG,enzyme sequences which provide a label for the polypeptide, andsequences which provide affinity probes, such as an antigen-antibodyrecognition.

[0044] The polypeptides of the invention are isolated and purified fromtissues and cell lines which express OPG binding protein, eitherextracted from lysates or from conditioned growth medium, and fromtransformed host cells expressing OPG binding protein. OPG bindingprotein may be obtained from murine myelomonocytic cell line 32-D (ATCCaccession no. CRL-11346). Human OPG binding protein, or nucleic acidsencoding same, may be isolated from human lymph node or fetal livertissue. Isolated OPG binding protein is free from association with humanproteins and other cell constituents.

[0045] A method for the purification of OPG binding protein from naturalsources (e.g. tissues and cell lines which normally express OPG bindingprotein) and from transfected host cells is also encompassed by theinvention. The purification process may employ one or more standardprotein purification steps in an appropriate order to obtain purifiedprotein. The chromatography steps can include ion exchange, gelfiltration, hydrophobic interaction, reverse phase, chromatofocusing,affinity chromatography employing an anti-OPG binding protein antibodyor biotin-streptavidin affinity complex and the like.

[0046] Antibodies

[0047] Antibodies specifically binding the polypeptides of the inventionare also encompassed by the invention. The antibodies may be produced byimmunization with full-length OPG binding protein, soluble forms of OPGbinding protein, or a fragment thereof. The antibodies of the inventionmay be polyclonal or monoclonal, or may be recombinant antibodies, suchas chimeric antibodies wherein the murine constant regions on light andheavy chains are replaced by human sequences, or CDR-grafted antibodieswherein only the complementary determining regions are of murine origin.Antibodies of the invention may also be human antibodies prepared, forexample, by immunization of transgenic animals capable of producinghuman antibodies (see, for example, PCT Application No. WO93/12227). Theantibodies are useful for detecting OPG binding protein in biologicalsamples, thereby allowing the identification of cells or tissues whichproduce the protein In addition, antibodies which bind to OPG bindingprotein and block interaction with other binding compounds may havetherapeutic use in modulating osteoclast differentiation and boneresorption.

[0048] Antibodies to the OPG binding protein may be useful in treatmentof bone diseases such as, osteoporosis and Paget's disease. Antibodiescan be tested for binding to the OPG binding protein in the absence orpresence of OPG and examined for their ability to inhibit ligand (OPGbinding protein) mediated osteoclastogenesis and/or bone resorption. Itis also anticipated that the peptides themselves may act as anantagonist of the ligand:receptor interaction and inhibitligand-mediated osteoclastogenesis, and peptides of the OPG bindingprotein will be explored for this purpose as well.

[0049] Compositions

[0050] The invention also provides for pharmaceutical compositionscomprising a therapeutically effective amount of the OPG binding proteinof the invention together with a pharmaceutically acceptable diluent,carrier, solubilizer, emulsifier, preservative and/or adjuvant. Theinvention also provides for pharmaceutical compositions comprising atherapeutically effective amount of an OPG binding protein agonist orantagonist. The term “therapeutically effective amount” means an amountwhich provides a therapeutic effect for a specified condition and routeof administration. The composition may be in a liquid or lyophilizedform and comprises a diluent (Tris, acetate or phosphate buffers) havingvarious pH values and ionic strengths, solubilizer such as Tween orPolysorbate, carriers such as human serum albumin or gelatin,preservatives such as thimerosal or benzyl alcohol, and antioxidantssuch as ascrobic acid or sodium metabisulfite. Selection of a particularcomposition will depend upon a number of factors, including thecondition being treated, the route of administration and thepharmacokinetic parameters desired. A more extensive survey of componentsuitable for pharmaceutical compositions is found in Remington'sPharmaceutical Sciences, 18th ed. A. R. Gennaro, ed. Mack, Easton, Pa.(1980).

[0051] In a preferred embodiment, compositions comprising soluble OPGbinding proteins are also provided. Also encompassed are compositionscomprising soluble OPG binding protein modified with water solublepolymers to increase solubility, stability, plasma half-life andbioavailability. Compositions may also comprise incorporation of solubleOPG binding protein into liposomes, microemulsions, micelles or vesiclesfor controlled delivery over an extended period of time. Soluble OPGbinding protein may be formulated into microparticles suitable forpulmonary administration.

[0052] Compositions of the invention may be administered by injection,either subcutaneous, intravenous or intramuscular, or by oral, nasal,pulmonary or rectal administration. The route of administrationeventually chosen will depend upon a number of factors and may beascertained by one skilled in the art.

[0053] The invention also provides for pharmaceutical compositionscomprising a therapeutically effective amount of the nucleic acids ofthe invention together with a pharmaceutically acceptable adjuvant.Nucleic acid compositions will be suitable for the delivery of part orall of the coding region of OPG binding protein and/or flanking regionsto cells and tissues as part of an anti-sense therapy regimen.

[0054] Methods of Use

[0055] OPG binding proteins may be used in a variety of assays fordetecting OPG and characterizing interactions with OPG. In general, theassay comprises incubating OPG binding protein with a biological samplecontaining OPG under conditions which permit binding to OPG to OPGbinding protein, and measuring the extent of binding. OPG may bepurified or present in mixtures, such as in body fluids or culturemedium. Assays may be developed which are qualitative or quantitative,with the latter being useful for determining the binding parameters(affinity constants and kinetics) of OPG to OPG binding protein and forquantitating levels of biologically active OPG in mixtures. Assays mayalso be used to evaluate the binding of OPG to fragments, analogs andderivatives of OPG binding protein and to identify new OPG and OPGbinding protein family members.

[0056] Binding of OPG to OPG binding protein may be carried out inseveral formats, including cell-based binding assays, membrane bindingassays, solution-phase assays and immunoassays. In general, trace levelsof labeled OPG are incubated with OPG binding protein samples for aspecified period of time followed by measurement of bound OPG byfiltration, electrochemiluminescent (ECL, ORIGEN system by IGEN),cell-based or immunoassays. Homogeneous assay technologies forradioactivity (SPA; Amersham) and time resolved fluoresence (HTRF,Packard) can also be implemented. Binding is detected by labeling OPG oran anti-OPG antibody with radioactive isotopes (125I, 35S, 3H),fluorescent dyes (fluorescein), lanthanide (Eu3+) chelates or cryptates,orbipyridyl-ruthenium (Ru2+) complexes. It is understood that the choiceof a labeled probe will depend upon the detection system used.Alternatively, OPG may be modified with an unlabled epitope tag (e.g.,biotin, peptides, His₆, myc) and bound to proteins such as streptavidin,anti-peptide or anti-protein antibodies which have a detectable label asdescribed above.

[0057] In an alternative method, OPG binding protein may be assayeddirectly using polyclonal or monoclonal antibodies to OPG bindingproteins in an immunoassay. Additional forms of OPG binding proteinscontaining epitope tags as described above may be used in solution andimmunoassays.

[0058] Methods for indentifying compounds which interact with OPGbinding protein are also encompassed by the invention. The methodcomprises incubating OPG binding protein with a compound underconditions which permit binding of the compound to OPG binding protein,and measuring the extent of binding. The compound may be substantiallypurified or present in a crude mixture. Binding compounds may be nucleicacids, proteins, peptides, carbohydrates, lipids or small molecularweight organic compounds. The compounds may be further characterized bytheir ability to increase or decrease OPG binding protein activity inorder to determine whether they act as an agonist or an antagonist.

[0059] OPG binding proteins are also useful for identification ofintracellular proteins which interact with the cytoplasmic domain by ayeast two-hybrid screening process. As an example, hybrid constructscomprising DNA encoding the N-terminal 50 amino acids of an OPG bindingprotein fused to a yeast GAL4-DNA binding domain may be used as atwo-hybrid bait plasmid. Positive clones emerging from the screening maybe characterized further to identify interacting proteins. Thisinformation may help elucidate a intracellular signaling mechanismassociated with OPG binding protein and provide intracellular targetsfor new drugs that modulate bone resorption.

[0060] OPG binding protein may be used to treat conditions characterizedby excessive bone density. The most common condition is osteopetrosis inwhich a genetic defect results in elevated bone mass and is usuallyfatal in the first few years of life. Osteopetrosis is preferablytreated by administration of soluble OPG binding protein.

[0061] The invention also encompasses modulators (agonists andantagonists) of OPG binding protein and the methods for obtaining them.An OPG binding protein modulator may either increase or decrease atleast one activity associated with OPG binding protein, such as abilityto bind OPG or some other interacting molecule or to regulate osteoclastmaturation. Typically, an agonist or antagonist may be a co-factor, suchas a protein, peptide, carbohydrate, lipid or small molecular weightmolecule, which interacts with OPG binding protein to regulate itsactivity. Potential polypeptide antagonists include antibodies whichreact with either soluble or membrane-associated forms of OPG bindingprotein, and soluble forms of OPG binding protein which comprise part orall of the extracellular domain of OPG binding protein. Molecules whichregulate OPG binding protein expression typically include nucleic acidswhich are complementary to nucleic acids encoding OPG binding proteinand which act as anti-sense regulators of expression.

[0062] OPG binding protein is involved in controlling formation ofmature osteoclasts, the primary cell type implicated in bone resorption.An increase in the rate of bone resorption (over that of bone formation)can lead to various bone disorders collectively referred to asosteopenias, and include osteoporosis, osteomyelitis, hypercalcemia,osteopenia brought on by surgery or steroid administration, Paget'sdisease, osteonecrosis, bone loss due to rheumatoid arthritis,periodontal bone loss, immobilization, prosthetic loosing and osteolyticmetastasis. Conversely, a decrease in the rate of bone resorption canlead to osteopetrosis, a condition marked by excessive bone density.Agonists and antagonists of OPG binding protein modulate osteoclastformation and may be administered to patients suffering from bonedisorders. Agonists and antagonists of OPG binding protein used for thetreatment of osteopenias may be administered alone or in combinationwith a therapeutically effective amount of a bone growth promoting agentincluding bone morphogenic factors designated BMP-1 to BMP-12,transforming growth factor-β and TGF-β family members, fibroblast growthfactors FGF-1 to FGF-10, interleukin-1 inhibitors, TNFα inhibitors,parathyroid hormone, E series prostaglandins, bisphosphonates andbone-enhancing minerals such as fluoride and calcium. Antagonists of OPGbinding proteins may be particularly useful in the treatment ofosteopenia.

[0063] The following examples are offered to more fully illustrate theinvention, but are not construed as limiting the scope thereof.

EXAMPLE 1 Identification of a Cell Line Source for an OPG BindingProtein

[0064] Osteoprotegerin (OPG) negatively regulates osteoclastogenesis invitro and in vivo. Since OPG is a TNFR-related protein, it is likely tointeract with a TNF-related family member while mediating its effects.With one exception, all known members of the TNF superfamily are type IItransmembrane proteins expressed on the cell surface. To identify asource of an OPG binding protein, recombinant OPG-Fc fusion proteinswere used as immunoprobes to screen for OPG binding proteins located onthe surface of various cell lines and primary hematopoietic cells.

[0065] Cell lines that grew as adherent cultures in vitro were treatedusing the following methods: Cells were plated into 24 well tissueculture plates (Falcon), then allowed to grow to approxiamtely 80%confluency. The growth media was then removed, and the adherent cultureswere washed with phosphate buffered saline (PBS) (Gibco) containing 1%fetal calf serum (FCS). Recombinant mouse OPG [22-194]-Fc and human OPG[22-201]-Fc fusion proteins (see U.S. Ser. No. 08/706,945 filed Sep. 3,1996) were individually diluted to 5 ug/ml in PBS containing 1% FCS,then added to the cultures and allowed to incubate for 45 min at 0° C.The OPG-Fc fusion protein solution was discarded, and the cells werewashed in PBS-FCS solution as described above. The cultures were thenexposed to phycoeyrthrin-conguated goat F(ab′) anti-human IgG secondaryantibody (Southern Biotechnology Associates Cat. # 2043-09) diluted intoPBS-FCS. After a 30-45 min incubation at 0° C., the solution wasdiscarded, and the cultures were washed as described above. The cellswere then analysed by immunofluorescent microscopy to detect cell lineswhich express a cell surface OPG binding protein.

[0066] Suspension cell cultures were analysed in a similar manner withthe following modifications: The diluent and wash buffer consisted ofcalcium- and magnesium-free phosphate buffered saline containing 1% FCS.Cells were harvested from exponentially replicating cultures in growthmedia, pelleted by centrifugation, then resuspended at 1×10⁷ cells/ml ina 96 well microtiter tissue culture plate (Falcon). Cells weresequentially exposed to recombinant OPG-Fc fusion proteins, thensecondary antibody as described above, and the cells were washed bycentrifugation between each step. The cells were then analysed byfluorescence activated cell sorting (FACS) using a Becton DickinsonFACscan.

[0067] Using this approach, the murine myelomonocytic cell line 32D(ATCC accession no. CRL-11346) was found to express a surface moleculewhich could be detected with both the mouse OPG[22-194]-Fc and the humanOPG[22-201]-Fc fusion proteins. Secondary antibody alone did not bind tothe surface of 32D cells nor did purified human IgG1 Fc, indicating thatbinding of the OPG-Fc fusion proteins was due to the OPG moiety. Thisbinding could be competed in a dose dependent manner by the addition ofrecombinant murine or human OPG[22-401] protein. Thus the OPG regionrequired for its biological activity is capable of specifically bindingto a 32D-derived surface molecule.

EXAMPLE 2 Expression Cloning of a Murine OPG Binding Protein

[0068] A cDNA library was prepared from 32D mRNA, and ligated into themammalian expression vector pcDNA3.1(+) (Invitrogen, San Diego, Calif.).Exponentially growing 32D cells maintained in the presence ofrecombinant interleukin-3 were harvested, and total cell RNA waspurified by acid guanidinium thiocyanate-phenol-chloroform extraction(Chomczynski and Sacchi. Anal. Biochem. 162, 156-159, (1987)). The poly(A+) mRNA fraction was obtained from the total RNA preparation byadsorption to, and elution from, Dynabeads Oligo (dT)25 (Dynal Corp)using the manufacturer's recommended procedures. A directional, oligo-dTprimed cDNA library was prepared using the Superscript Plasmid System(Gibco BRL, Gaithersburg, Md.) using the manufacturer's recommendedprocedures. The resulting cDNA was digested to completion with Sal I andNot I restriction endonuclease, then fractionated by size exclusion gelchromatography. The highest molecular weight fractions were selected,and then ligated into the polyliker region of the plasmid vectorpcDNA3.1(+) (Invitrogen, San Diego, Calif.). This vector contains theCMV promotor upstream of multiple cloning site, and directs high levelexpression in eukaryotic cells. The library was then electroporated intocompetent E. coli (ElectroMAX DH10B, Gibco, N.Y.), and titered on LBagar containing 100 ug/ml ampicillin. The library was then arrayed intosegregated pools containing approximately 1000 clones/pool, and 1.0 mlcultures of each pool were grown for 16-20 hr at 37° C. Plasmid DNA fromeach culture was prepared using the Qiagen Qiawell 96 Ultra Plasmid Kit(catalog #16191) following manufacturer's recommended procedures.

[0069] Arrayed pools of 32D cDNA expression library were individuallylipofected into COS-7 cultures, then assayed for the acquisition of acell surface OPG binding protein. To do this, COS-7 cells were plated ata density of 1×10⁶ per ml in six-well tissue culture plates (Costar),then cultured overnight in DMEM (Gibco) containing 10% FCS.Approximately 2 μg of plasmid DNA from each pool was diluted into 0.5 mlof serum-free DMEM, then sterilized by centrifugation through a 0.2 μmSpin-X column (Costar). Simultaneously, 10 μl of Lipofectamine (LifeTechnologies Cat # 18324-012) was added to a separate tube containing0.5 ml of serum-free DMEM. The DNA and Lipofectamine solutions weremixed, and allowed to incubate at RT for 30 min. The COS-7 cell cultureswere then washed with serum-free DMEM, and the DNA-lipofectaminecomplexes were exposed to the cultures for 2-5 hr at 37° C. After thisperiod, the media was removed, and replaced with DMEM containing 10%FCS.The cells were then cultured for 48 hr at 37° C.

[0070] To detect cultures that express an OPG binding protein, thegrowth media was removed, and the cells were washed with PBS-FCSsolution. A 1.0 ml volume of PBS-FCS containing 5 μg/ml of humanOPG[22-201]-Fc fusion protein was added to each well and incubated at RTfor 1 hr. The cells were washed three times with PBS-FCS solution, andthen fixed in PBS containing 2% paraformaldehyde and 0.2% glutaraldehydein PBS at RT for 5 min. The cultures were washed once with PBS-FCS, thenincubated for 1 hr at 65° C. while immersed in PBS-FCS solution. Thecultures were allowed to cool, and the PBS-FCS solution was aspirated.The cultures were then incubated with an alkaline-phosphatase conjugatedgoat anti-human IgG (Fc specific) antibody (SIGMA Product # A-9544) atRt for 30 min, then washed three-times with 20 mM Tris-Cl (pH 7.6), and137 mM NaCl. Immune complexes that formed during these steps weredetected by assaying for alkaline phosphatase activity using the FastRed TR/AS-MX Substrate Kit (Pierce, Cat. # 34034) following themanufacturer's recommended procedures.

[0071] Using this approach, a total of approximately 300,000 independent32D cDNA clones were screened, represented by 300 transfected pools of1000 clones each. A single well was identifed that contained cells whichacquired the ability to be specifically decorated by the OPG-Fc fusionprotein. This pool was subdivided by sequential rounds of sib selection,yeilding a single plasmid clone 32D-F3 (FIG. 1). 32D-F3 plasmid DNA wasthen transfected into COS-7 cells, which were immunostained with eitherFITC-conjugated goat anti-human IgG secondary antibody alone, humanOPG[22-201]-Fc fusion protein plus secondary, or with ATAR-Fc fusionprotein (ATAR also known as HVEM; Montgomery et al. Cell 87, 427-436(1996)) (FIG. 2). The secondary antibody alone did not bind toCOS-7/32D-F3 cells, nor did the ATAR-Fc fusion protein. Only the OPG Fcfusion protein bound to the COS-7/32D-F3 cells, indicating that 32D-F3encoded an OPG binding protein displayed on the surface of expressingcells.

EXAMPLE 3 OPG Binding Protein Sequence

[0072] The 32D-F3 clone isolated above contained an approximately 2.3 kbcDNA insert (FIG. 1), which was sequenced in both directions on anApplied Biosystems 373A automated DNA sequencer using primer-driven Taqdye-terminator reactions (Applied Biosystems) following themanufacturer's recommended procedures. The resulting nucleotide sequenceobtained was compared to the DNA sequence database using the FASTAprogram (GCG, Univeristy of Wisconsin), and analysed for the presence oflong open reading frames (LORF's) using the “Six-way open reading frame”application (Frames) (GCG, Univeristy of Wisconsin). A LORF of 316 aminoacid (aa) residues beginning at methionine was detected in theappropriate orientation, and was preceded by a 5′ untranslated region ofabout 150 bp. The 5′ untranslated region contained an in-frame stopcodon upstream of the predicted start codon. This indicates that thestructure of the 32D-F3 plasmid is consistent with its ability toutilize the CMV promotor region to direct expression of a 316 aa geneproduct in mammalian cells.

[0073] The predicted OPG binding protein sequence was then compared tothe existing database of known protein sequences using a modifiedversion of the FASTA program (Pearson, Meth. Enzymol. 183, 63-98(1990)). The amino acid sequence was also analysed for the presence ofspecific motifs conserved in all known members of the tumor necrosisfactor (TNF) superfamily using the sequence profile method of (Gribskovet al. Proc. Natl. Acad. Sci. USA 83, 4355-4359 (1987)), as modified byLüethy et al. Protein Sci. 3, 139-146 (1994)). There appeared to besignificant homology throughout the OPG binding protein to severalmembers of the TNF superfamily. The mouse OPG binding protein appear tobe most closely related to the mouse and human homologs of both TRAILand CD40 ligand. Further analysis of the OPG binding protein sequenceindicated a strong match to the TNF superfamily, with a highlysignificant Z score of 19.46.

[0074] The OPG binding protein amino acid sequence contains a probablehydrophobic transmembrane domain that begins at a M49 and extends toL69. Based on this configuration relative to the methionine start codon,the OPG binding protein is predicted to be a type II transmembraneprotein, with a short N-terminal intracellular domain, and a longerC-terminal extracellular domain (FIG. 4). This would be similar to allknown TNF family members, with the exception of lymphotoxin alpha(Nagata and Golstein, Science 267, 1449-1456 (1995)).

EXAMPLE 4 Expression of Human OPG Binding Protein mRNA

[0075] Multiple human tissue northern blots (Clontech, Palo Alto,Calif.) were probed with a ³²P-dCTP labelled 32D-F3 restriction fragmentto detect the size of the human transcript and to determine patterns ofexpression. Northern blots were prehybridized in 5× SSPE, 50% formamide,5× Denhardt's solution, 0.5% SDS, and 100 μg/ml denatured salmon spermDNA for 2-4 hr at 42° C. The blots were then hybridized in 5× SSPE, 50%formamide, 2× Denhardt's solution, 0.1% SDS, 100 μg/ml denatured salmonsperm DNA, and 5 ng/ml labelled probe for 18-24 hr at 42° C. The blotswere then washed in 2× SSC for 10 min at RT, 1× SSC for 10 min at 50°C., then in 0.5× SSC for 10-15 min.

[0076] Using a probe derived from the mouse cDNA and hybridization understringent conditions, a predominant mRNA species with a relativemolecular mass of about 2.5 kb was detected in lymph nodes (FIG. 3). Afaint signal was also detected at the same relative molecular mass infetal liver mRNA. No OPG binding protein transcripts were detected inthe other tissues examined. The data suggest that expression of OPGbinding protein mRNA was extremely restricted in human tissues. The dataalso indicate that the cDNA clone isolated is very close to the size ofthe native transcript, suggesting 32D-F3 is a full length clone.

EXAMPLE 5 Molecular Cloning of the Human OPG Binding Protein

[0077] The human homolog of the OPG binding protein is expressed as anapproximately 2.5 kb mRNA in human peripheral lymph nodes and isdetected by hybridization with a mouse cDNA probe under stringenthybdization conditions. DNA encoding human OPG binding protein isobtained by screening a human lymph node cDNA library by eitherrecombinant bacteriphage plaque, or transformed bacterial colony,hybridiziation methods (Sambrook et al. Molecular Cloning: A LaboratoryManual Cold Spring Harbor Press, New York (1989)). To this the phage orplasmid cDNA library are screened using radioactively-labeled probesderived from the murine OPG binding protein clone 32D-F3. The probes areused to screen nitrocellulose filter lifted from a plated library. Thesefilters are prehybridized and then hybridized using conditions specifiedin Example 4, ultimately giving rise to purified clones of the human OPGbinding protein cDNA. Inserts obtained from any human OPG bindingprotein clones would be sequenced and analysed as described in Example3.

[0078] A human lymph node poly A+ RNA (Clontech, Inc., Palo Alto,Calif.) was analysed for the presence of OPG-bp transcripts aspreviously in U.S. Ser. No. 08/577,788, filed Dec. 22, 1995. A northernblot of this RNA sample probed under stringent conditions with a32P-labeled mouse OPG-bp probe indicated the presence of human OPG-bptranscripts. An oligo dT-primed cDNA library was then synthesized fromthe lymph node mRNA using the SuperScript kit (GIBCO life Technologies,Gaithersberg, Md.) as described in example 2. The resulting cDNA wassize selected, and the high molecular fraction ligated to plasmid vectorpcDNA 3.1 (+) (Invitrogen, San Diego, Calif.). Electrocompetent E. coliDH10 (GIBCO life Technologies, Gaithersberg, Md.) were transformed, and1×10⁶ ampicillin resistant transformants were screened by colonyhybridization using a 32P-labeled mouse OPG binding protein probe.

[0079] A plasmid clone of putative human OPG binding protein cDNA wasisolated, phuOPGbp-1.1, and contained a 2.3 kp insert. The resultingnucleotide sequence of the phuOPGbp-1.1 insert was approximately 80-85%homologous to the mouse OPG binding protein cDNA sequence. Translationof the insert DNA sequence indicated the presence of a long open readingframe predicted to encode a 317 aa polypeptide (FIG. 4). Comparison ofthe mouse and human OPG-bp polypeptides shows that they are ˜87%identical, indicating that this protein is highly conserved duringevolution.

[0080] The human OPG binding protein DNA and protein sequences were notpresent in Genbank, and there were no homologus EST sequences. As withthe murine homolog, the human OPG binding protein shows strong sequencesimilarity to all members of the TNFα superfamily of cytokines.

EXAMPLE 6 Cloning and Bacterial Expression of OPG Binding Protein

[0081] PCR amplification employing the primer pairs and templatesdescribed below are used to generate various forms of murine OPG bindingproteins. One primer of each pair introduces a TAA stop codon and aunique XhoI or SacII site following the carboxy terminus of the gene.The other primer of each pair introduces a unique NdeI site, aN-terminal methionine, and optimized codons for the amino terminalportion of the gene. PCR and thermocycling is performed using standardrecombinant DNA methodology. The PCR products are purified, restrictiondigested, and inserted into the unique NdeI and XhoI or SacII sites ofvector pAMG21 (ATCC accession no. 98113) and transformed into theprototrophic E. coli 393 or 2596. Other commonly used E. coli expressionvectors and host cells are also suitable for expression. Aftertransformation, the clones are selected, plasmid DNA is isolated and thesequence of the OPG binding protein insert is confirmed.

[0082] pAMG21-Murine OPG Binding Protein F75-3161

[0083] This construct was engineered to be 242 amino acids in length andhave the following N-terminal and C-terminal residues, NH₂-Met (75)-Asp-Pro-Asn-Arg-------Gln-Asp-Ile-Asp(316)—COOH. The template to beused for PCR was pcDNA/32D-F3 and oligonucleotides #1581-72 and #1581-76were the primer pair to be used for PCR and cloning this gene construct.1581-72: (SEQ ID NO:_)5′-GTTCTCCTCATATGGATCCAAACCGTATTTCTGAAGACAGCACTCAC TGCTT-3′ 1581-76:(SEQ ID NO:_) 5′-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3′

[0084] pAMG21-Murine OPG Binding Protein [95-316]

[0085] This construct was engineered to be 223 amino acids in length andhave the following N-terminal and C-terminal residues, NH₂-Met-His(95)-Glu-Asn-Ala-Gly-------Gln-Asp-Ile-Asp(316)—COOH. The template usedfor PCR was pcDNA/32D-F3 and oligonucleotides #1591-90 and #1591-95 werethe primer pair used for PCR and cloning this gene construct. 1591-90:(SEQ ID NO:_) 5′-ATTTGATTCTAGAAGGAGGAATAACATATGCATGAAAACGCAGGTCT GCAG-3′1591-95: (SEQ ID NO:_) 5′-TATCCGCGGATCCTCGAGTTAGTCTATGTCCTGAACTTTGAA-3′

[0086] pAMG21-Murine OPG Binding Protein [107-316]

[0087] This construct was engineered to be 211 amino acids in length andhave the following N-terminal and C-terminal residues,NH₂-Met-Ser(107)-Glu-Asp-Thr-Leu-------Gln-Asp-Ile-Asp(316)—COOH. Thetemplate used for PCR was pcDNA/32D-F3 and oligonucleotides #1591-93 and#1591-95 were the primer pair used for PCR and cloning this geneconstruct. 1591-93: (SEQ ID NO:_)5′-ATTTGATTCTAGAAGGAGGAATAACATATGTCTGAAGACACTCTGCC GGACTCC-3′ 1591-95:(SEQ ID NO:_) 5′-TATCCGCGGATCCTCGAGTTAGTCTATGTCCTGAACTTTGAA-3′

[0088] pAMG21-Murine OPG Binding Protein [118-316]

[0089] This construct was engineered to be 199 amino acids in length andhave the following N-terminal and C-terminal residues,NH₂-Met(118)-Lys-Gln-Ala-Phe-Gln-------Gln-Asp-Ile-Asp(316)—COOH. Thetemplate used for PCR was pcDNA/32D-F3 and oligonucleotides #1591-94 and#1591-95 were the primer pair used for PCR and cloning this geneconstruct. 1591-94: (SEQ ID NO:_)5′-ATTTGATTCTAGAAGGAGGAATAACATATGAAACAAGCTTTTCAGGG G-3′ 1591-95: (SEQ IDNO:_) 5′-TATCCGCGGATCCTCGAGTTAGTCTATGTCCTGAACTTTGAA-3′

[0090] pAMG21-Murine OPG binding protein [128-316]

[0091] This construct was engineered to be 190 amino acids in length andhave the following N-terminal and C-terminal residues,NH₂-Met-Lys(128)-Glu-Leu-Gln-His - - - Gln-Asp-Ile-Asp(316)—COOH. Thetemplate used for PCR was pcDNA/32D-F3 and oligonucleotides #1591-91 and#1591-95 were the primer pair used for PCR and cloning this geneconstruct. 1591-91: (SEQ ID NO:_)5′-ATTTGATTCTAGAAGGAGGAATAACATATGAAAGAACTGCAGCACAT TGTG-3′ 1591-95: (SEQID NO:_) 5′TATCCGCGGATCCTCGAGTTAGTCTATGTCCTGAACTTTGAA-3′

[0092] pAMG21-Murine OPG Binding Protein [137-316]

[0093] This construct was engineered to be 181 amino acids in length andhave the following N-terminal and C-terminal residues,NH₂-Met-Gln(137)-Arg-Phe-Ser-Gly-------Gln-Asp-Ile-Asp(316)—COOH. Thetemplate used for PCR was pcDNA/32D-F3 and oligonucleotides #1591-92 and#1591-95 were the primer pair used for PCR and cloning this geneconstruct. 1591-92: (SEQ ID NO:_)5′-ATTTGATTCTAGAAGGAGGAATAACATATGCAGCGTTTCTCTGGTGC TCCA-3′ 1591-95: (SEQID NO:_) 5′-TATCCGCGGATCCTCGAGTTAGTCTATGTCCTGAACTTTGAA-3′

[0094] pAMG21-Murine OPG Binding Protein [146-316]

[0095] This construct is engineered to be 171 amino acids in length andhave the following N-terminal and C-terminal residues,NH₂-Met(146)-Glu-Gly-Ser-Trp-------Gln-Asp-Ile-Asp(316)—COOH. Thetemplate to be used for PCR is pAMG21-murine OPG binding protein[75-316] described above and oligonucleotides #1600-98 and #1581-76 willbe the primer pair to be used for PCR and cloning this gene construct.1600-98: (SEQ ID NO:_) 5′-GTTCTCCTCATATGGAAGGTTCTTGGTTGGATGTGGCCCA-3′1581-76: (SEQ ID NO:_) 5′-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3′

[0096] pAMG21-Murine OPG Binding Protein [156-316]

[0097] This construct is engineered to be 162 amino acids in length andhave the following N-terminal and C-terminal residues,NH₂-Met-Arg(156)-Gly-Lys-Pro--------Gln-Asp-Ile-Asp(316)—COOH. Thetemplate to be used for PCR is pAMG21-murine OPG binding protein[158-316] below and oligonucleotides #1619-86 and #1581-76 will be theprimer pair to be used for PCR and cloning this gene construct. 1619-86:(SEQ ID NO:_) 5′-GTTCTCCTCATATGCGTGGTAAACCTGAAGCTCAACCATTTGCA-3′1581-76: (SEQ ID NO:_) 5′-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3′

[0098] pAMG21-Murine OPG Binding Protein [158-316]

[0099] This construct was engineered to be 160 amino acids in length andhave the following N-terminal and C-terminal residues,NH₂-Met-Lys(158)-Pro-Glu-Ala--------Gln-Asp-Ile-Asp(316)—COOH. Thetemplate to be used for PCR was pcDNA/32D-F3 and oligonucleotides#1581-73 and #1581-76 were the primer pair to be used for PCR andcloning this gene construct. 1581-73: (SEQ ID NO:_)5′-GTTCTCCTCATATGAAACCTGAAGCTCAACCATTTGCACACCTCACC ATCAAT-3′ 1581-76:(SEQ ID NO:_) 5′-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3′

[0100] pAMG21-Murine OPG Binding Protein [166-316]

[0101] This construct is engineered to be 152 amino acids in length andhave the following N-terminal and C-terminal residues,NH₂-Met-His(166)-Leu-Thr-Ile--------Gln-Asp-Ile-Asp(316)—COOH. Thetemplate to be used for PCR is pcDNA/32D-F3 and oligonucleotides#1581-75 and #1581-76 will be the primer pair to be used for PCR andcloning this gene construct. 1581-75: (SEQ ID NO:_)5′-GTTCTCCTCATATGCATTTAACTATTAACGCTGCATCTATCCCATCG GGTTCCCATAAAGTCACT-3′1581-76: (SEQ ID NO:_) 5′-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3′

[0102] pAMG21-Murine OPG Binding Protein [168-316]

[0103] This construct is engineered to be 150 amino acids in length andhave the following N-terminal and C-terminal residues,NH₂-Met-Thr(168)-Ile-Asn-Ala--------Gln-Asp-Ile-Asp(316)—COOH. Thetemplate to be used for PCR is pcDNA/32D-F3 and oligonucleotides#1581-74 and #1581-76 will be the primer pair to be used for PCR andcloning. 1581-74: (SEQ ID NO:  )5′-GTTCTCCTCATATGACTATTAACGCTGCATCTATCCCATCGGGTTCC CATAAAGTCACT-3′1581-76: (SEQ ID NO:  ) 5′-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3′

[0104] It is understood that the above constructs are examples and oneskilled in the art may readily obtain other forms of OPG binding proteinusing the general methodology presented her.

[0105] Recombinant bacterial constructs pAMG21-murine OPG bindingprotein [75-316], [95-316], [107-316], [118-316], [128-316], [137-316],and [158-316] have been cloned, DNA sequence confirmed, and levels ofrecombinant gene product expression following induction has beenexamined. All constructs produced levels of recombinant gene productwhich was readily visible following SDS polyacrylamide gelelectrophoresis and coomassie staining of crude lysates. Growth oftransformed E. coli 393 or 2596, induction of OPG binding proteinexpression and isolation of inclusion bodies containing OPG bindingprotein is done according to procedures described in U.S. Ser. No.08/577,788 filed Dec. 22, 1995. Purification of OPG binding proteinsfrom inclusion bodies requires solubilization and renaturing of OPGbinding protein using procedures available to one skilled in the art.Recombinant murine OPG binding protein [158-316] was found to beproduced mostly insolubly, but about 40% was found in the solublefraction. Recombinant protein was purified from the soluble fraction asdescribed below and its bioactivity examined.

EXAMPLE 7 Purification of Recombinant Murine OPG Ligand [158-316]

[0106] Frozen bacterial cells harboring expressed murine OPG bindingprotein (158-316) were thawed and resuspended in 20 mM tris-HCl pH 7.0,10 mM EDTA. The cell suspension (20% w/v) was then homogenized by threepasses through a microfluidizer. The lysed cell suspension wascentrifuged in a JA14 rotor at 10,000 rpm for 45 minutes. SDS-PAGEanalysis showed a band of approximately 18kd molecular weight present inboth inclusion bodies and the supernatant. The soluble fraction was thenapplied to a Pharmacia SP Sepharose 4FF column equilibrated with 10 mMMES pH 6.0. The OPG binding protein was eluted with a 20 column volumegradient of 0-0.4M NaCl in MES pH 6.0. Fractions containing OPG bindingprotein were then applied to an ABX Bakerbond column equilibrated with20 mM MES pH 6.0. OPG binding protein was eluted with a 15CV gradient of0-0.5M NaCl in MES pH 6.0. The final product was over 95% homogeneous bySDS-PAGE. N-terminal sequencing gave the following sequence:Met-Lys-Pro-Glu-Ala-Gln-Pro-Phe-Ala-His which was identified to thatpredicted for a polypeptide starting at residue 158 (with an initiatormethionine). The relative molecular weight of the protein duringSDS-PAGE does not change upon reduction.

EXAMPLE 8 In vitro Bioactivity of Recombinant Soluble OPG-bp

[0107] Recombinant OPG protein has previously been shown to blockvitamin D3-dependent osteoclast formation from bone marrow and spleenprecursors in an osteoclast forming assay as described in U.S. Ser. No.08/577,788. Since OPG binding protein binds to OPG, and is a novelmember of the TNF family of ligands, it is a potential target of OPGbioactivity. Recombinant soluble OPG binding protein (158-316),representing the minimal core TNFα-like domain, was tested for itsability to modulate osteoclast differentiation from osteoclastprecursors. Bone marrow cells were isolated from adult mouse femurs, andtreated with M-CSF. The non-adherent fraction was co-cultured with ST2cells in the presence and absence of both vitamin D3 and dexamethasone.As previously shown, osteoclasts develop only from co-culturescontaining stromal cells (ST2), vitamin D3 and dexamethasone.Recombinant soluble OPG binding protein was added at varyingconcentrations ranging from 0.16 to 500 ng/ml and osteoclast maturationwas determined by TRAP solution assay and by visual observation. OPGbinding protein strongly stimulated osteoclast differentiation andmaturation in a dose dependent manner, with half-maximal effects in the1-2 ng/ml range, suggesting that it acts as an potent inducer ofosteoclastogenesis in vitro (FIG. 5). The effect of OPG binding proteinis blocked by recombinant OPG (FIG. 6).

[0108] To test whether OPG binding protein could replace the stroma andadded steroids, cultures were established using M-CSF at varyingconcentrations to promote the growth of osteoclast precursors andvarious amounts of OPG binding protein were also added. As shown in FIG.6, OPG binding protein dose dependently stimultated TRAP activity, andthe magnitude of the stimulation was dependent on the level of addedM-CSF suggesting that these two factors together are pivotal forosteoclast development. To confirm the biological relevance of this lastobservation, cultures were established on bovine cortical bone slicesand the effects of M-CSF and OPG binding protein either alone ortogether were tested. As shown in FIG. 7, OPG binding protein in thepresence of M-CSF stimulated the formation of large TRAP positiveosteoclasts that eroded the bone surface resulting in pits. Thus, OPGbinding protein acts as an osteoclastogenesis stimulating(differentiation) factor. This suggests that OPG blocks osteoclastdevelopment by sequestering OPG binding protein.

EXAMPLE 9 In vivo Activity of Recombinant Soluble OPG Binding Protein

[0109] Based on in vitro studies, recombinant murine OPG binding protein[158-316] produced in E. coli is a potent inducer of osteoclastdevelopment from myeloid precursors. To determine its effects in vivo,male BDF1 mice aged 4-5 weeks (Charles River Laboratories) receivedsubcutaneous injections of OPG binding protein [158-316] twice a day forthree days and on the morning of the fourth day (days 0, 1, 2, and 3).Five groups of mice (n=4) received carrier alone, or 1, 5, 25 or 100μg/of of OPG binding protein [158-316] per day. An additional 5 groupsof mice (n=4) received the above doses of carrier or of OPG bindingprotein [158-316] and in addition received human Fc-OPG [22-194] at 1mg/Kg/day (approximately 20 μg/day) by single daily subcutaneousinjection. Whole blood ionized calcium was determined prior to treatmenton day 0 and 3-4 hours after the first daily injection of of OPG bindingprotein [158-316] on days 1, 2, and 3. Four hours after the lastinjection on day 3 the mice were sacrificed and radiographs were taken.

[0110] Recombinant of OPG binding protein [158-316] produced asignificant increase in blood ionized calcium after two days of treamentat dose of 5 μg/day and higher (FIG. 8). The severity of thehypercalcemia indicates a potent induction of osteoclast activityresulting from increased bone resorption. Concurrent OPG administrationlimited hypercalcemia at doses of OPG binding protein [158-316] of 5 and25 μg/day, but not at 100 μg/day. These same animal were analysed byradiaography to determine if there were any effects on bone mineraldensity visible by X-ray (FIG. 9). Recombinant of OPG binding protein[158-316] injected for 3 days decreased bone density in the proximaltibia of mice in a dose-dependent manner. The reduction in bone densitywas particularly evident in mice receiving 100 μg/d confirming that theprofound hypercalcemia in these animals was produced from increased boneresorption and the resulting release of calcium from the skeleton. Thesedata clearly indicate that of OPG binding protein [158-316] acts in vivoto promote bone resorption, leading to systemic hypercalcemia, andrecombinant OPG abbrogates these effects.

EXAMPLE 10 Cloning and Expression of Soluble OPG Binding Protein inMammalian Cells

[0111] The full length clone of murine and human OPG binding protein canbe expressed in mammalian cells as previously described in Example 2.Alternatively, the cDNA clones can be modified to encode secreted formsof the protein when expressed in mammalian cells. To do this, thenatural 5′end of the cDNA encoding the intiation codon, and extendingapproximately through the first 69 amino acid of the protein, inludingthe transmembrane spanning region, could be replaced with a signalpeptide leader sequence. For example, DNA sequences encoding theinitiation codon and signal peptide of a known gene can be spliced tothe OPG binding protein cDNA sequence beginning anywhere after theregion encoding amino acid residue 68. The resulting recombinant clonesare predicted to produce secreted forms of OPB binding protein inmammalian cells, and should undergo post translational modificationswhich normally occur in the C-terminal extracellular domain of OPGbinding protein, such as glycoslyation. Using this strategy, a secretedform of OPG binding protein was constructed which has at its 5′ end themurine OPG signal peptide, and at its 3′ end the human IgG1 Fc domain.The plasmid vector pCEP4/muOPG[22-401]-Fc as described in U.S. Ser. No.08/577,788, filed Dec. 22, 1995, was digested with NotI to cleavebetween the 3′ end of OPG and the Fc gene. The linearized DNA was thenpartially digested with XmnI to cleave only between residues 23 and 24of OPG leaving a blunt end. The restriction digests were thendephosphorylated with CIP and the vector portion of this digest(including residues 1-23 of OPG and Fc) was gel purified.

[0112] The murine OPG binding protein cDNA region encoding amino acidreisudes 69-316 were PCR amplified using Pfu Polymerase (Stratagene, SanDiego, Calif.) from the plasmid template using primers the followingoligonucleotides:

[0113] 1602-61: CCT CTA GGC CTG TAC TTT CGA GCG CAG ATG

[0114] 1602-59: CCT CTG CGG CCG CGT CTA TGT CCT GAA CTT TG

[0115] The 1602-61 oligonucleotide amplifies the 5′ end of the gene andcontains an artificial an StuI site. The 1602-59 primer amplifies the 3′end of the gene and contians an artifical NotI site. The resulting PCRproduct obtained was digested with NotI and StuI, then gel purified. Thepurified PCR product was ligated with vector, then used to transformelectrocompetent E. coli DH10B cells. The resulting clone was sequencedto confirm the intergrity of the amplified sequence and restriction sitejunctions. This plasmid was then used to transfect human 293fibroblasts, and the OPG binding protein-Fc fusion protein was collectedform culture media as previously described in U.S. Ser. No. 08/577,788,filed Dec. 22, 1995.

[0116] Using a similar strategy, an expression vector was designed thatis capable of expressing a N-terminal truncation of fused to the humanIgG1 Fc domain. This construct consists of the murine OPG signal peptide(aa residue 1-21), fused in frame to murine OPG binding protein residues158-316, followed by an inframe fusion to human IgG1 Fc domain. To dothis, the plasmid vector pCEP4/murine OPG [22-401] (U.S. Ser. No.08/577,788, filed Dec. 22, 1995), was digested with HindIII and NotI toremove the entire OPG reading frame. Murine OPG binding protein,residues 158-316 were PCR amplified using from the plasmid templatepCDNA/32D-F3 using the following primers:

[0117] 1616-44: CCT CTC TCG AGT GGA CAA CCC AGA AGC CTG AGG CCC AGC CATTTG C

[0118] 1602-59: CCT CTG CGG CCG CGT CTA TGT CCT GAA CTT TG

[0119] 1616-44 amplifies OPG binding protein starting at residue 158 aswell as containing residues 16-21 of the muOPG signal peptide with anartificial XhoI site. 1602-59 amplifies the 3′ end of the gene and addsan in-frame NotI site. The PCR product was digested with NotI and XhoIand then gel purified.

[0120] The Follwing complimentary primers were annealed to eachother toform an adapter encoding the murine OPG signal peptide and Kozaksequence surrounding the translation initiation site: 1616-41: AGC TTCCAC CAT GAA CAA GTG GCT GTG CTG CGC ACT CCT GGT GCT CCT GGA CAT CA1616-42: TCG ATG ATG TCC AGG AGC ACC AGG AGT GCG CAG CAC AGC CAC TTG TTCATG GTG GA

[0121] These primers were annealed, generating 5′ overhangs compatiblewith HindIII on the 5′ end and XhoI on the 3′ end. The digested vectorobtianed above, the annealed oligos, and the digested PCR fragment wereligated together and electroporated into DH10B cells. The resultingclone was sequenced to confirm authentic reconstruction of the junctionbetween the signal peptide, OPG binding protein fragment encodingresidues 158-316, and the IgG1 Fc domain. The recombiant plasmid waspurified, transfected into human 293 fibroblasts, and expressed as aconditioned media product as described above.

EXAMPLE 11 Peptides of the OPG Binding Protein and Preparation ofPolyclonal and Monoclonal Antibodies to the Protein

[0122] Antibodies to specific regions of the OPG binding protein may beobtained by immunization with peptides from OPG binding protein. Thesepeptides may be used alone, or conjugated forms of the peptide may beused for immunization.

[0123] The crystal structure of mature TNFα has been described [E. Y.Jones, D. I. Stuart, and N. P. C. Walker (1990) J. Cell Sci. Suppl. 13,11-18] and the monomer forms an antiparallel β-pleated sheet sandwichwith a jellyroll topology. Ten antiparallel β-strands are observed inthis crystal structure and form a beta sandwich with one beta sheetconsisting of strands B′BIDG and the other of strands C′CHEF [E. Y.Jones et al., ibid.] Two loops of mature TNFα have been implicated frommutagenesis studies to make contacts with receptor, these being theloops formed between beta strand B & B′ and the loop between betastrands E & F [C. R. Goh, C-S. Loh, and A. G. Porter (1991) ProteinEngineering 4, 785-791]. The crystal structure of the complex formedbetween TNFβ and the extracellular domain of the 55 kd TNF receptor(TNF-R55) has been solved and the receptor-ligand contacts have beendescribed [D. W. Banner, A. D'Arcy, W. Janes, R. Gentz, H-J. Schoenfeld,C. Broger, H. Loetscher, and W. Lesslauer (1993) Cell 73, 431-445]. Inagreement with mutagenesis studies described above [C. R. Goh et al.,ibid.] the corresponding loops BB′ and EF of the ligand TNFβ were foundto make the majority of contacts with the receptor in the resolvedcrystal structure of the TNFb:TNF-R55 complex. The amino acid sequenceof murine OPG binding protein was compared to the amino acid sequencesof TNFα and TNFβ. The regions of murine OPG binding proteincorresponding to the BB′ and EF loops were predicted based on thiscomparison and peptides have been designed and are described below

[0124] A. Antigen(s): Recombinant murine OPG binding protein [158-316]has been used as an antigen (ag) for immunization of animals asdescribed below, and serum will be examined using approaches describedbelow. Peptides to the putative BB′ and EF loops of murine OPG bindingprotein have been synthesized and will be used for immunization; thesepeptides are: BB′ loop peptide: NH2--NAASIPSGSHKVTLSSWYHDRGWAKIS--COOHBB′ loop-Cys peptide: NH2--NAASIPSGSHKVTLSSWYHDRGWAKISC--COOH EF looppeptide: NH2--VYVVKTSIKIPSSHNLM--COOH EF loop-Cys peptide:NH2--VYVVKTSIKIPSSHNLMC--COOH

[0125] Peptides with a carboxy-terminal cysteine residue have been usedfor conjugation using approaches described in section B below, and havebeen used for immunization.

[0126] B. Keyhole Limpet Hemocyanin or Bovine Serum Albumin Conjugation:Selected peptides or protein fragments may be conjugated to keyholelimpet hemocyanin (KLH) in order to increase their immunogenicity inanimals. Also, bovine serum albumin (BSA) conjugated peptides or proteinfragments may be utilized in the EIA protocol. Imject MaleimideActivated KLH or BSA (Pierce Chemical Company, Rockford, Ill.) isreconstituted in dH₂O to a final concentration of 10 mg/ml. Peptide orprotein fragments are dissolved in phosphate buffer then mixed with anequivalent mass (g/g) of KLH or BSA. The conjugation is allowed to reactfor 2 hours at room temperature (rt) with gentle stirring. The solutionis then passed over a desalting column or dialyzed against PBSovernight. The peptide conjugate is stored at −20° C. until used inimmunizations or in EIAs.

[0127] C. Immunization: Balb/c mice, (Charles Rivers Laboratories,Wilmington, Mass.) Lou rats, or New Zealand White rabbits will besubcutaneously injected (SQI) with ag (50 μg, 150 μg, and 100 μgrespectively) emulsified in Complete Freund's Adjuvant (CFA, 50%vol/vol; Difco Laboratories, Detroit, Mich.). Rabbits are then boostedtwo or three times at 2 week intervals with antigen prepared in similarfashion in Incomplete Freund's Adjuvant (ICFA; Difco Laboratories,Detroit, Mich.). Mice and rats are boosted approximately every 4 weeks.Seven days following the second boost, test bleeds are performed andserum antibody titers determined. When a titer has developed in rabbits,weekly production bleeds of 50 mls are taken for 6 consecutive weeks.Mice and rats are selected for hybridoma production based on serum titerlevels; animals with half-maximal titers greater than 5000 are used.Adjustments to this protocol may be applied by one skilled in the art;for example, various types of immunomodulators are now available and maybe incorporated into this protocol.

[0128] D. Enzyme-linked Immunosorbent Assay (EIA): EIAs will beperformed to determine serum antibody (ab) titres of individual animals,and later for the screening of potential hybridomas. Flat bottom,high-binding, 96-well microtitration EIA/RIA plates (Costar Corporation,Cambridge, Mass.) will be coated with purified recombinant protein orprotein fragment (antigen, ag) at 5 μg per ml in carbonate-bicarbonatebuffer, pH 9.2 (0.015 M Na₂CO₃, 0.035 M NaHCO₃). Protein fragments maybe conjugated to bovine serum albumin (BSA) if necessary. Fifty μl of agwill be added to each well. Plates will then be covered with acetatefilm (ICN Biomedicals, Inc., Costa Mesa, Calif.) and incubated at roomtemperature (rt) on a rocking platform for 2 hours or over-night at 4°C. Plates will be blocked for 30 minutes at rt with 250 μl per well 5%BSA solution prepared by mixing 1 part BSA diluent/blocking solutionconcentrate (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, Md.)with 1 part deionized water (dH₂O). Blocking solution having beendiscarded, 50 μl of serum 2-fold dilutions (1:100 through 1:12,800) orhybridoma tissue culture supernatants will be added to each well. Serumdiluent is 1% BSA (10% BSA diluent/blocking solution concentrate diluted1:10 in Dulbecco's Phosphate Buffered Saline, D-PBS; Gibco BRL, GrandIsland, N.Y.)) while hybridoma supernatants are tested undiluted. In thecase of hybridoma screening, one well is maintained as a conjugatecontrol, and a second well as a positive ab control. Plates are againincubated at rt, rocking for 1 hour, then washed 4 times using a 1×preparation of wash solution 20× concentrate (Kirkegaard and PerryLaboratories, Inc., Gaithersburg, Md.) in dH₂O. Horseradish peroxidaseconjugated secondary ab (Boeringer Mannheim Biochemicals, Indianapolis,Ind.) diluted in 1% BSA is then incubated in each well for 30 minutes.Plates are washed as before, blotted dry, and ABTS peroxidase singlecomponent substrate (Kirkegaard and Perry Laboratories, Inc.,Gaithersburg, Md.) is added. Absorbance is read at 405 nm for each wellusing a Microplate EL310 reader (Bio-tek Instruments, Inc., Winooski,Vt.). Half-maximal titre of serum antibody is calculated by plotting thelog₁₀ of the serum dilution versus the optical density at 405, thenextrapolating at the 50% point of the maximal optical density obtainedby that serum. Hybridomas are selected as positive if optical densityscores greater than 5-fold above background. Adjustments to thisprotocol may be applied; in example, conjugated secondary antibody maybe chosen for specificity or non-cross-reactivity.

[0129] E. Cell fusion: The animal selected for hybridoma production isintravenously injected with 50 to 100 μg of ag in PBS. Four days later,the animal is sacrificed by carbon dioxide and its spleen collectedunder sterile conditions into 35 ml Dulbeccos' Modified Eagle's Mediumcontaining 200 U/ml Penicillin G, 200 μg/ml Streptomycin Sulfate, and 4mM glutamine (2× P/S/G DMEM). The spleen is trimmed of excess fattytissue, then rinsed through 4 dishes of clean 2× P/S/G DMEM. It is nexttransferred to a sterile stomacher bag (Tekmar, Cincinnati, Ohio)containing 10 ml of 2× P/S/G DMEM and disrupted to single cellsuspension with the Stomacher Lab Blender 80 (Seward Laboratory UACHouse; London, England). As cells are released from the spleen capsuleinto the media, they are removed from the bag and transferred to asterile 50 ml conical centrifuge tube (Becton Dickinson and Company,Lincoln Park, N.J.). Fresh media is added to the bag and the process iscontinued until the entire cell content of the spleen is released. Thesesplenocytes are washed 3 times by centrifugation at 225×g for 10minutes.

[0130] Concurrently, log phase cultures of myeloma cells, Sp2/0-Ag14 orY3-Ag1.2.3 for mouse or rat splenocyte fusions, respectively, (AmericanType Culture Collection; Rockville, Md.) grown in complete medium (DMEM,10% inactivated fetal bovine serum, 2 mM glutamine, 0.1 mM non-essentialamino acids, 1 mM sodium pyruvate, and 10 mM hepes buffer; GibcoLaboratories, Grand Island, N.Y.) are washed in similar fashion. Thesplenocytes are combined with the myeloma cells and pelleted once again.The media is aspirated from the cell pellet and 2 ml of polyethyleneglycol 1500 (PEG 1500; Boehringer Mannheim Biochemicals, Indianapolis,Ind.) is gently mixed into the cells over the course of 1 minute.Thereafter, an equal volume of 2× P/S/G DMEM is slowly added. The cellsare allowed to fuse at 37° C. for 2 minutes, then an additional 6 ml of2× P/S/G DMEM is added. The cells are again set at 37° C. for 3 minutes.Finally, 35 ml of 2× P/S/G DMEM is added to the cell suspension, and thecells pelleted by centrifugation. Media is aspirated from the pellet andthe cells gently resuspended in complete medium. The cells aredistributed over 96-well flat-bottom tissue culture plates (BectonDickinson Labware; Lincoln Park, N.J.) by single drops from a 5 mlpipette. Plates are incubated overnight in humidified conditions at 37°C. 5% CO₂. The next day, an equal volume of selection medium is added toeach well. Selection consists of 0.1 mM hypoxanthine, 4×10⁻⁴ mMaminopterin, and 1.6×10⁻² mM thymidine in complete medium. The fusionplates are incubated for 7 days followed by 2 changes of medium duringthe next 3 days; HAT selection medium is used after each fluid change.Tissue culture supernatants are taken 3 to 4 days after the last fluidchange from each hybrid-containing well and tested by EIA for specificantibody reactivity. This protocol has been modified by that in Hudsonand Hay, “Practical Immunology, Second Edition”, Blackwell ScientificPublications.

[0131] While the present invention has been described in terms of thepreferred embodiments, it is understood that variations andmodifications will occur to those skilled in the art. Therefore, it isintended that the appended claims cover all such equivalent variationswhich come within the scope of the invention as claimed.

What is claimed is:
 1. An isolated nucleic acid encoding anosteoprotegerin binding protein selected from the group consisting of:a) the nucleic acid sequence as in FIG. 1 (SEQ ID NO:______) and FIG. 4(SEQ ID NO:______); b) nucleic acids which hybridize to the polypeptidecoding regions as shown in FIG. 1 (SEQ ID NO:______) and FIG. 4 (SEQ IDNO:______) and remain hybridized under high stringency conditions; andc) nucleic acids which are degenerate to the nucleic acids of (a) or(b).
 2. The nucleic acid of claim 1 which is cDNA, genomic DNA,synthetic DNA or RNA.
 3. A polypeptide encoded by the nucleic acid ofclaim
 1. 4. The nucleic acid of claim 1 including one or more codonspreferred for Escherichia coli expression.
 5. The nucleic acid of claim1 having a detectable label attached thereto.
 6. A nucleic acid encodinga polypeptide comprising the amino acid sequence of residues 1-316 andresidues 70-316 as shown in FIG. 1 (SEQ ID NO:______).
 7. A nucleic acidencoding a polypeptide comprising amino acid sequence of residues 1-317and residues 69-317 as shown in FIG. 4 (SEQ ID NO:______);
 8. A nucleicacid encoding a soluble osteoprotegerin binding protein.
 9. The nucleicacid of claim 8 encoding a polypeptide comprising residues 69-317 asshown in FIG. 4 (SEQ ID NO:______) and truncations thereof;
 10. Anexpression vector comprising the nucleic acid of claims 1 and
 9. 11. Theexpression vector of claim 10 wherein the nucleic acid comprises thepolypeptide-encoding region as shown in FIG. 1 (SEQ ID NO:______) andFIG. 4 (SEQ ID NO:______);
 12. A host cell transformed or transfectedwith the expression vector of claim
 10. 13. The host cell of claim 12which is a eucaryotic or procaryotic cell.
 14. The host cell of claim 13which is Escherichia coli.
 15. A process for the production of anosteoprotegerin binding protein comprising: growing under suitablenutrient conditions host cells transformed or transfected with thenucleic acid of claim 1; and isolating the polypeptide product of theexpression of the nucleic acid.
 16. A polypeptide produced by theprocess of claim
 15. 17. A purified and isolated osteoprotegerin bindingprotein, or fragment, analog, or derivative thereof.
 18. The protein ofclaim 17 which is a human osteoprotegerin.
 19. The protein of claim 17having the amino acid sequence as shown in FIG. 1 (SEQ ID NO:______) andFIG. 4 (SEQ ID NO:______).
 20. The protein of claim 17 which has beencovalently modified with a water-soluble polymer.
 21. The protein ofclaim 20 wherein the polymer is polyethylene glycol.
 22. The protein ofclaim 17 which is a soluble osteoprotegerin binding protein.
 23. Theprotein of claim 22 comprising the amino acid sequence from residues70-316 inclusive as shown in FIG. 1 (SEQ ID NO:______), or a fragment,analog, or derivative thereof.
 24. The protein of claim 22 comprisingthe amino acid sequence from residues 69-317 inclusive as shown in FIG.4 (SEQ ID NO:______) and truncations thereof.
 25. An antibody orfragment thereof which specifically binds an osteoprotegerin bindingprotein.
 26. The antibody of claim 25 which is a monoclonal antibody.27. A method for detecting the presence of an osteoprotegerin bindingprotein in a biological sample comprising: incubating the sample withthe antibody of claim 25 under conditions that allow binding of theantibody to the osteoprotegerin binding protein; and detecting the boundantibody.
 28. A method for detecting the presence of osteoprotegerin ina biological sample comprising: incubating the sample with anosteoprotegerin binding protein under conditions that allow binding ofthe protein to osteoprotegerin; and measuring the bound osteoprotegerinbinding protein.
 29. A method to assess the ability of a candidatecompound to bind to an osteoprotegerin binding protein comprising:incubating the osteoprotegerin binding protein with the candidatecompound under conditions that allow binding; and measuring the boundcompound.
 30. The method of claim 29 wherein the compound is an agonistor an antagonist of an osteoprotegerin binding protein.
 31. A method ofregulating expression of an osteoprotegerin binding protein in an animalcomprising administering to the animal a nucleic acid complementary tothe nucleic acids as shown in FIG. 1 (SEQ ID NO:______) and FIG. 4 (SEQID NO:______).
 32. A pharmaceutical composition comprising atherapeutically effective amount of an osteoprotegerin binding proteinin a pharmaceutically acceptable carrier, adjuvant, solubilizer,stabilizer and/or anti-oxidant.
 33. The composition of claim 32 whereinthe osteoprotegerin binding protein is a human osteoprotegerin bindingprotein.
 34. A method of treating bone disease in a mammal comprisingadministering a therapeutically effective amount of a modulator of anosteoprotegerin binding protein.
 35. The method of claim 34 wherein themodulator is a soluble form of an osteoprotegerin binding protein. 36.The method of claim 35 wherein the modulator is an antibody, or fragmentthereof, which specifically binds an osteoprotegerin binding protein.